Real time PCR info - (Oct/24/2009 )

Hello,
I have some questions about Real time RT PCR as i am doing it for the first time. Although i have read few articles on this topic but still i feel that I am confused with the following terms and their usage:

1.Normalisation and when we use it
2. Standard curve and why we use this
3. Housekeeping genes
4. Difference between Normalisation and standard curve

Any help in the above mentioned query will be highly appreciated.
I am trying to see the splicing mechanism of a certain gene construct.

Thanks anii

-anii-

anii on Oct 25 2009, 07:08 AM said:

Hello,
I have some questions about Real time RT PCR as i am doing it for the first time. Although i have read few articles on this topic but still i feel that I am confused with the following terms and their usage:

1.Normalisation and when we use it
2. Standard curve and why we use this
3. Housekeeping genes
4. Difference between Normalisation and standard curve

Any help in the above mentioned query will be highly appreciated.
I am trying to see the splicing mechanism of a certain gene construct.

Thanks anii

1) Normalisation is the correction in the background signal (Usually done with a reference dye ROX) but nowadays systems which do not need to use this dye have come into the market!!!
2) Standard curve is used for quantitation of your product (in amount or copy number or any other unit). A line graph is one of the standard curve (y=mx+c)
3) housekeeping gene is typically a constitutive gene that is transcribed at a relatively constant level. The housekeeping gene's products are typically needed for maintenance of the cell. Housekeeping genes are used as internal standards in quantitative polymerase chain reaction since it is generally assumed that their expression is unaffected by experimental conditions (from wikipedia)
4) You must know it by now!!!

-Pradeep Iyer-

1) Normalization can mean more things, please specify the context. Normalization can also mean normalising your gene of interest (GOI) to a housekeeping gene, that is used in relative quantification. You compare the changes in ROI to the changes of the housekeeping gene to account for the fluctuation in the starting amount of RNA in your RT reaction. Look up this tutorial for the three methods commonly used to normalise, standard curve method, delta-delta Ct method (most used) and Pfaffl method (efficiency normalized delta-delta). This may also answer your question 4.

-Trof-

thanks everyone. Your inputs really helped me.

-anii-

anii on Oct 28 2009, 10:19 PM said:

thanks everyone. Your inputs really helped me.

you re welcome.. that is why we al are ehre for.. helping each other!!!